Diabetic retinopathy, a leading cause of blindness, is characterized by blood-retinal barrier breakdown from endothelial cell damage. The causes of retinal vascular damage in diabetes are incompletely understood and current treatments fail to address the underlying pathophysiology. The specific objective of this proposal is to identify the mechanism(s) through which diabetes impairs blood-retinal barrier integrity. The investigators have shown that vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), high glucose and histamine reduce tight junction protein expression in retinal capillary endothelial cells, and VEGF increases tyrosine phosphorylation of ZO-1. VEGF expression is increased in early diabetic retinopathy and is associated with vascular leakage. Therefore, the general hypothesis is proposed that blood-retinal barrier breakdown in diabetes results from VEGF acting on endothelial cell tight junctions. The rationale for this project is that an understanding of the regulation of tight junction proteins may permit the design of a means to prevent abnormal permeability and microvascular damage. To this end, three specific hypotheses will be tested: (1) that VEGF regulates tight junction protein content and phosphorylation; (2) that VEGF increases permeability through tight junctions in retinal microvascular endothelial cells; and (3) that VEGF disturbs retinal vascular tight junctions in vivo. These hypotheses will be investigated by: (1) quantification of ZO-1 and occludin protein and mRNA content and stability, phosphorylation, and ZO-1-occludin protein-protein interactions in bovine retinal capillary endothelial cells; (2) determination of water and albumin permeability across endothelial cell monolayers under varying exposures to VEGF, hyperglycemia, and hydrostatic pressure; and (3) analysis of retinal tight junction proteins in eyes of VEGF-injected normal rats, and of diabetic rats. This combination of molecular and functional analyses will provide new insights into the pathogenesis of vascular permeability in diabetes and other retinopathies.